Chemical constituents from leaves and trunk bark of Rinorea oblongifolia (Violaceae)

Abstract Two new coruleoellagic acid derivatives, 3,4′,5,5′,-tetramethylcoruleoellagic acid (1); 3′,4,4′,5,5′-pentamethylcoruleoellagic acid (2) and a new friedelane-type triterpene derivative rinol (5), were isolated from leaves and trunk bark of Rinorea oblongifolia (Violaceae) along with seven known compounds including 3,3′,4,4′,5′-pentamethylcoruleoellagic acid (3), hexamethylcoruleoellagic acid (4), 28-hydroxyfriedelin (6), friedelin (7), friedelan-3-ol (8), scopoletin (9) and β-sitosterol-3-O-β-D-glucopyranoside (10). Their structures were elucidated by means of spectroscopic methods including IR, 1D and 2D NMR in conjunction with mass spectrometry. Crude extracts of leaves and trunk bark as well as compounds 1–4 were evaluated for their antibacterial activities against 7 pathogenic bacterial strains (Streptococcus pneumoniae ATCC49619, Staphylococcus aureus ATCC 43300, Klepsiella pneumoniae ATCC 700603, Haemophilus influenza ATCC 49247, Escherichia coli ATCC 25922, Pseudomonas aeruginosa HM601, Staphylococcus aureus BAA 977). Compound (3) displayed noteworthy activity against Haemophilus influenza with MIC value of 9.38 µg/mL. Graphical Abstract


Introduction
Rinorea oblongifolia (C.H.Wright) is one of the tallest species of the genus which could reach 12 m in height and 30 cm in diameter (Burkill 1985). In Africa, the leaves extract is used as an expectorant; it is also used to cure fever and aches. The root bark infusion is good for constipation, stomach aches, rheumatismal aches and oedema (Agnaniet et al. 2003). Previous phytochemical investigation of others species of same family revealed the presence of a wide range of secondary metabolites, including ellagic acids, alkaloids (Chen and Aisa 2017), glycosinolates (Montaut et al. 2017) and flavonoids (Qin et al. 2016;stewart et al. 2000), some of which showed interesting biological properties, such as antioxidant, anti-inflammatory and antimalarial activities (Dinesh et al. 2011). In our ongoing search for bioactive secondary metabolites from Cameroonian Rinorea species, bioassay-guided isolation of the active constituents of leaves and trunk bark of Rinorea oblongifolia was carried out. As results, four coruleoellagic acid derivatives (1-4), including two new ones (1 and 2), and four friedelan-type triterpenes (5-8) one of which is new (5), together with two other known compounds (9 and 10) were isolated. In this paper, we wish to describe the isolation and structural elucidation of these compounds as well as the evaluation of the antibacterial activities of some of them.
Compound 1 was obtained as a yellow lemon amorphous powder and reacted positively to the FeCl 3 reagent, suggesting its phenolic nature. Its molecular formula C 18 H 14 O 10 indicating 12 degree of unsaturation, was assigned from its HR-ESI-TOF mass spectrum which showed in negative mode the pseudo molecular ion peak [M-H] þ at m/z 389.0516. The FT-IR spectrum of this compound displayed vibration bands for free and chelated hydroxyl groups (3419 and 3282 cm À1 ), and conjugated carbonyl (1741 cm À1 ). We also observed carbon double bond (C ¼ C) of olefinic and aromatic ring (1453 cm À1 ) and ether function (1224 cm À1 ). The UV spectrum showed absorption bands at k max 210, 248 and 378 nm characteristic of chromophore of ellagic acid derivative (Atta-Ur-Rahman et al. 2001). Broad band proton decoupled carbon 13 C spectrum (Table S1) displayed sixteen signals instead of eighteen as indicated in the molecular formula, suggesting the presence of two isochrones carbons. These signals were sorted by DEPT spectra into four methoxyl groups at d C 62.0; 61.9; 61.8 and 60.8.
The remaining twelve signals were assigned to quaternary carbons including one carbonyl lactone at d C 158.3, seven sp2 oxygenated carbons at d C 155. 2, 152.6, 146.4, 142.0, 140.0, 128.8, 128.0, and four non-oxygenated sp2 carbon at d C , 113.2, 112.0, 111.6 and 107.5. The 1 H NMR spectrum of this compound showed four three protons singlets at d H 4.13, 4.02, 3.92, and 3.89 which correlated on HSQC spectrum with carbon at d C ; 61.8, 60.8; 61.9 and 62.0, respectively. In agreement with the molecular formula, it remains two phenolic groups which did not appear on the proton NMR spectrum, due to the fact that, this spectrum was recorded in the mixture of DMSO-d 6 and MeOD-d 4 , which caused the exchange of acidic proton and the disappearance of their signals . According to the molecular formula and the phenolic nature of compound 1, the presence of two hydroxyl groups in its structure was confirmed by chemical evidence: methylation of compound 1, carried out by refluxing it in acetone in the presence of methyl iodide and potassium carbonate, led to a derivative identified as hexamethylcoruleoellagic acid (4). The positions of the four methoxyls and the two hydroxyl groups in 1 were therefore established as follow: two of the four methoxyl groups (d H 3.92, and 3.89) were located to adjacent position from the correlation observed between these two groups on the NOESY spectrum ( Figure S32). The third methoxyl group resonating at d H 4.13, was located at C-3 position from the comparison of its chemical shift with the one of the methoxyl occupying similar position in the structure of 3,3 0 ,4,4 0 ,5 0 -pentamethylcoruleoellagic acid (Lakornwong et al. 2018). The fact that this third methoxyl group did not show any correlation with the fourth methoxyl group at d H 4.02, suggested that this latter methoxyl group is located at C-5 position. Therefore the two phenolic groups were located to the remaining two free positions C-4 and C-3 0 , respectively. Thus, the structure assigned to compound 1, was established as 3,4 0 ,5,5 0 ,-tetramethylcoruleoellagic acid.
Compound 2 was obtained as a yellowish amorphous powder, and reacted positively to FeCl 3 reagent. Its high-resolution EI-mass spectrum showed a molecular ion [M þ ] at m/z 404.0743 in agreement with empirical formula C 19 H 16 O 10 (calculated 404.0744). The IR spectrum of this compound showed vibration bands at 3420 (br, OH), 1743, 1722 (lactone carbonyl), 2940, 2854 and 1073 (-OMe) and 1591, 1473, 1453 (aromatic). Acetylation of this compound using acetic anhydride in pyridine at room temperature for twelve hours, yielded a monoacetylated derivative which acetoxy signal appeared at d H 2.01 on its 1 H NMR spectrum, this result indicated that this compound possesses one free phenolic group. Analysis of 13 C and 1 H NMR spectral data of compound 2 (Table S1) combined to its UV spectrum which showed absorption band at at k max 244, 248 and 378 nm revealed that compound 2 has ellagic-type skeleton, similar to that of compound 1. In addition, the presence of five methoxyl groups in compound 2 was inferred by five three protons singlets at d H 3.95; 3.96; 3.99; 4.16 and 4.20 which correlated on HSQC spectrum with carbon at d C 62.3, 62.2, 61.8 and 62.4, respectively. Thus compare to compound 1, compound 2 possesses five methoxyl and one phenolic groups. It remains to position on the two tri-substituted aromatic rings of coruleoellagic acid derivative those six substituents. Three of the five methoxyl groups were logically located on one aromatic ring at positions C-3 0 , C-4 0 and C-5 0 . The two remaining others and the phenolic group were located on the second aromatic ring. The fact that the chemical shift of one of the methoxyl group was deshielded (d H 4.20) indicated that methoxyl group was at peri-postion to carbonyl (position 5). Furthermore, its correlation on the NOESY spectrum ( Figure S33) with another methoxyl group at d H 4.16 suggests that they were close spatially. This led to locate the second methoxyl group at position 4 and a hydroxyl group at position 3. From the above spectroscopic data, structure 2 was assigned to this compound trivially named 3 0 ,4,4 0 ,5,5 0 -pentamethylcoruleoellagic acid.
Compound 5 was obtained as white powder from hexane: EtOAc (3:1), ½a 24 D -54.2 (c ¼ 0.00241, MeOH). It reacted positively with Libermann-Buchard test suggesting its triterpenoidic nature. Its molecular formula, C 30 H 50 O 2 , corresponding to 6 double bond equivalents was deduced from its FAB-MS spectrum which showed in positive mode the pseudo-molecular ion peak [M þ H] þ at m/z 443.3. The IR spectrum of 5 exhibited vibration bands for hydroxyl group (3475 cm À1 ) and ether function (1174 cm À1 ). The 13 C NMR spectrum displayed 30 signals corresponding to the thirty carbons figuring in the molecular formula. The analysis of these signals by DEPT technics revealed the presence of seven methyl at d C 8. 6, 16.4, 16.4, 18.8, 31.8, 32.2 and 35.0; twelve sp3 methylene at d C 19.6, 20.5, 30.4, 32.7, 33.2, 34.1, 35.1, 35.5, 36.3, 39.2, 39.4 and 73.1 among which one oxymethylene (d C 73.1); and four methines all hybridized sp3 at d C 43.0, 52.8, 54.0 and 57.4 (Table S2). This result led to the conclusion that this compound possesses seven quaternary carbons, all hybridized sp3 among which one hemiacetal at d C 106.4. The methyl signal at d C 8.6 which correlated on HSQC spectrum with proton at d H 1.23 which appeared on the 1 H NMR spectrum was characteristic of carbon C-23 of the friedelane type skeleton (Mahato and kundu 1994). Were also observed on the 1 H NMR spectrum of this compound six three protons singlets corresponding to six angular methyl groups. An AB spin system appearing as two doublets at d H 3.27 (1H; d, J ¼ 8 Hz) and d H 4.28 (1H; d, J ¼ 8 Hz) which correlated on the HSQC spectrum with same carbon at d C 73.1 was inferred to the diastereotopic protons attributed to an oxymethylene group. In agreement with the number of unsaturation, the sixth double bond equivalent was attributed to a pyranne ring, established between carbon C-24 and C-3, resulting from a reaction between a primary alcohol at C-24 position with a keton function located at C-3. This was confirmed by HMBC ( Figure S34) correlation observed between proton H-4 at d H 1.59 with carbon C-23 (d C 8.6), C-24 (d C 73.1) and C-3 (d C 106.4). The relative stereochemistry of the OH group of the hemiacetal was established to be at equatorial position from the correlation observed on the NOESY spectrum ( Figure S35) between proton H-24b (d H 4.27) with the methyls at C-23 and C-25 and proton H-24a (d H 3.74) with proton H-1 ( Figure S35). From the above spectroscopic data, structure 5 was assigned to the compound which corresponds to a 24-hydroxy-3-oxohemiacetalfriedelane to which trivially named rinol was given.
Crude extracts of leaves and trunk bark as well as some of pure compounds (1-4) have been evaluated for their antibacterial potency against a wide range of microorganisms including Streptococcus pneumoniae ATCC49619, Staphylococcus aureus ATCC 43300, Klepsiella pneumoniae ATCC 700603, Haemophilus influenza ATCC 49247, Escherichia coli ATCC 25922, Pseudomonas aeruginosa HM601, Staphylococcus aureus BAA 977. The results showed that six of seven tested bacteria were not sensitive to those extract except Haemophilus influenza ATCC 49247 which was sensitive at 500 mg/ mL. Concerning the pure compounds, only compound 1 (3 0 ,4,4 0 ,5,5 0 -pentamethylcoruleoellagic acid) and compound 3 (3,3 0 ,4,4 0 ,5 0 -pentamethylcoruleoellagic acid) displayed a broad-spectrum of action compare to other compounds; compound 3 being the most active against Haemophilus influenza ATCC 49247 with a MIC ranged from 9.38 to 50 mg/mL (Table S3).

General experimental procedures
Melting points (mp) were performed on a Buchi-535 melting point apparatus and are uncorrected. UV spectra were measured on a Hitachi U-3200 Spectrophotometer. IR spectra were recorded on a Shimadgu 8900 FT-IR spectrophotometer in KBr disks. The NMR spectra in DMSO-d6, acetone-d6 and pyridine-d 5 were obtained using Bruker Av-300, Av-500, Avance-500 Cryo-Probe and AV-III-HD 800 Cryo-Probe instruments, operating at 300, 500 and 800 MHz for 1 H NMR and 75, 125 and 200 MHz for 13 C NMR. Chemical shifts are given in d (ppm) using tetramethylsilane (TMS) as internal standard. EI-MS, HR-EI-MS, HR-ESI-MS and Fast atom bombardment mass spectra (FAB-MS) were obtained with a JEOL JMS-600H mass spectrometer. Analytical thin-layer chromatography (TLC) was performed on precoated silica gel plates (Merck 60F254; 20 Â 20, 0.25 mm). Column chromatography was carry out using silica gel (70-230 mesh; Merck). Chromatograms were visualized by spraying with a solution of 10% H 2 SO 4 or under ultraviolet light of wavelength 254 and 366 nm.

Plant material
The different parts of plant, consist of leaves and trunk bark were collected from Kala Mountain, Centre region, Cameroon in August 2017. The plant was authenticated by M. Mgansop, plant taxonomist at the National Herbarium of Cameroon, where a voucher specimen was deposited under the No. 20037 SRF/CAM.

Extraction and isolation
Air-dried trunk bark (2.0 kg) of Rinorea oblongifolia were powdered and extracted by maceration (10 L x 3) with the mixture of CH 2 Cl 2 /MeOH (1:1) at room temperature for two days. After filtration, the organic solution was concentrated in vacuo to yield a viscous crude extract (98 g). This extract was solubilized in the mixture of MeOH/H 2 O (9:1) and submitted to successive liquid-liquid partition in order to obtain n-Hexane (21g), CH 2 Cl 2 (30 g), EtOAc (19 g) and n-BuOH (17g) sub-fractions.
Methylation: In a 50 mL two-neck, round bottom flask, placed in an ice bath, containing compound 1 (3.5 mg) and K 2 CO 3 (6 mg) dissolved in a 5 mL of acetone, was added dropwise the methyl iodide (2 mL) over a period of 10 minutes. The mixture was stirred for overnight at room temperature. After reaction completion, the solvent was evaporated to give crude residue. This residue was diluted with water (20 mL) and extracted three times with chloroform (3x15 mL). The combined chloroform fractions were dried (MgSO 4 ) and evaporated. The residue was purified by chromatography, eluting with Hex/EtOAc (3:1) to give compound 4 (2.6 mg, 69.4%).
Antibacterial assay: The minimum inhibitory concentrations (MICs) of extracts and compounds were determined according to the Clinical Laboratory Standards Institute M07-A9 microdilution method using 96-wells microtitre plates (CLSI, 2012). Seven human pathogenic bacteria (Streptococcus pneumoniae ATCC49619, Staphylococcus aureus ATCC 43300, Klepsiella pneumoniae ATCC 700603, Haemophilus influenza ATCC 49247, Escherichia coli ATCC 25922, Pseudomonas aeruginosa HM601, Staphylococcus aureus BAA 977) obtained from BEI resources and the American Type Culture Collection were used for the test. 100 mL of two-fold diluted extracts/compounds and reference drugs in Muller Hinton Broth (Sigma Aldrich) were added to the wells, followed by addition of 100 mL of bacteria inoculum standardized at 1.5 Â 10 6 cells/mL. A blank column was included for sterility control. The concentrations of extracts and tested compounds ranged from 31.25 mg/mL to 500 mg/mL, from 3.125 to 50 mg/mL and that of Ciprofloxacin ranged from 0.5 mg/mL to 64 mg/mL. After 24 hours of incubation at 37 C, the turbidity was observed as an indication of growth. MIC was defined as the lowest concentration inhibiting the visible growth of bacteria. All tests were performed in triplicate.